ASYMMETRIC STRUCTURE OF PROTHROMBIN (FACTOR II): CALCIUM AND LIPID BINDING SITES, AND CARBOHYDRATE DISTRIBUTION Item Type text; Dissertation-Reproduction (electronic) Authors Benson, Bradley Jonnell, 1945- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 10/10/2021 08:15:19 Link to Item http://hdl.handle.net/10150/290366 INFORMATION TO USERS This material was produced from a microfilm copy of the original document. While the most advanced technological means to photograph and reproduce this document have been used, the quality is heavily dependent upon the quality of the original submitted. 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The University of Arizona, Ph.D., 1974 Chemistry, biological Xerox University Microfilms, Ann Arbor, Michigan 48106 THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. ASYMMETRIC STRUCTURE OF PROTHROMBIN (FACTOR II): CALCIUM AND LIPID BINDING SITES, AND CARBOHYDRATE DISTRIBUTION by Bradley Jonnell Benson A Dissertation Submitted to the Faculty of the COMMITTEE ON BIOCHEMISTRY (GRADUATE) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 19 7 4 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE I hereby recommend that this dissertation prepared under my direction by Bradley Jonnell Benson . entitled Asymmetric Structure of Prothrombin (Factor II): Calcium and Lipid Binding Sites, and Carbohydrate Distribution be accepted as fulfilling the dissertation requirement of the degree of Doctor of Philosophy (<1?*/ Dissertation Director Date ' After inspection of the final copy of the dissertation, the follo\;ing members of the Final Examination Committee concur in its approval and recommend its acceptance:- 1/7 > •>/>/?/ a/# Ch(Mtl£ 1 / This approval and acceptance is contingent on the candidate's adequate performance and defense of this dissertation at the final oral examination. The inclusion of this sheet bound into the library copy of the dissertation is evidence of satisfactory performance at the final examination. STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduc­ tion of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the interest of schol­ arship. In all other instances, however, permission must be obtained from the author. SIGNED: ACKNOWLEDGMENTS It would be impossible to thank or credit individually all those who contributed to this endeavor. I must however, acknowledge the help and advice of the following people. Professor Donald J. Hanahan in whose laboratory and under whose patient guidance and encouragement this work was carried out. Dr. Michael A. Wells and Dr. Walter Kisiel whose helpful criti­ cisms and invaluable discussions are deeply appreciated. Jamie Noel, J. Scott Hayes, C. G. Crawford, Katie Smith, and Thomas Allgyer, for whose many and varied discussion are sincerely appreciated. Mr. Ben Lee, whose excellent technical assistance during various aspects of this dissertation was very helpful. My parents whose support both moral and financial during this time is sincerely appreciated. The author was supported as a trainee on a Biochemistry trainee- ship from the National Institute of General Medical Sciences and on funds from the American Heart Association. iii TABLE OF CONTENTS Page LIST OF ILLUSTRATIONS vi LIST OF TABLES ix ABSTRACT x 1. INTRODUCTION 1 Overview of Blood Coagulation 1 Review of Other Preparations of Prothrombin and Their Physico-Chemical Properties 5 Role of Calcium Ions and Phospholipids in Blood Coagulation 14 Conversion of Prothrombin to Thrombin 19 Rationale 25 2. MATERIALS AND METHODS 27 Materials. 27 Methods 29 Assay Procedures 29 Preparative Methods 36 3. RESULTS 50 Purification of Bovine Factor II 50 Physico-Chemical Properties of Bovine Factor II 56 Molecular Weight Estimations 58 Extinction Coefficient of Bovine Factor II 63 Amino Acid and Carbohydrate Composition of Bovine Factor II 65 Isolation and Characterization of Conversion Polypeptides 68 Calcium Ion Binding of Prothrombin and Conversion Polypeptides 73 Phospholipid Binding of Prothrombin, Fragment 1 and Intermediate 1 87 Isolation and Characterization of the Calcium Binding and Carbohydrate Containing Peptides in Fragment 1 92 4. DISCUSSION 118 iv V TABLE OF CONTENTS--Continued Page Purification of Bovine Factor II 118 Physico-Chemical Properties of Bovine Factor II 121 Amino Acid and Carbohydrate Composition 124 Intermediates and Fragments of Prothrombin Conversion 125 REFERENCES 143 LIST OF ILLUSTRATIONS Figure Page 1. Cascade or waterfall scheme of the reactions of blood coagulation 3 2. Formation of complexes for two key reactions in blood coagulation 6 3. The conversion reaction of prothrombin to thrombin according to Heldebrant et al. (1973) 21 4. The prothrombin conversion pathways proposed by Stenn and Blout (1972) 22 5. Flow diagram of the purification of bovine prothrombin 40 6. Flow diagram for the generation and isolation of certain tryptic peptides from fragment 1 46 7. Elution profile of the barium citrate eluate on DEAE cellulose 52 8. Preparative electropherogram of prothrombin after DEAE cellulose chromatography 54 9. Analytical disc gel electrophoresis of the fractions obtained after preparative electrophoresis 55 10. Examination of purified prothrombin by disc and SDS electrophoresis 57 11. Standard curve for the estimation of the molecular weight of prothrombin by SDS electrophoresis 59 12. Plot of the logarithm of the concentration of factor II versus the square of the radial distance during low speed sedimentation equilibrium ultracentrifugation 61 13. Molecular weight standard curve of prothrombin from columns of Sephadex G-150 62 vi vii LIST OF ILLUSTRATIONS--Continued Figure Page 14. Standard curve for the estimation of the molecular weight of prothrombin by gel filtration in Bio-Gel P-200 64 15. Elution profile of thrombin on SE-Sephadex C-50 69 16. Preparative disc gel electropherogram of the isolation of fragment 1 and intermediate 1 71 17. SDS electrophoresis of prothrombin, intermediate 1, and fragment 1 72 18. Plot of calcium ion binding by factor II on gel filtration columns 77 19. Plot of the reversibility of calcium ion binding of prothrombin on Sephadex gel filtration columns 78 20. Plot of calcium ion binding by fragment 1 on gel filtration columns 80 21. Plot of calcium ion binding by intermediate 1 on gel filtration columns 81 22. Scatchard plot of the calcium ion binding to prothrombin and fragment 1 82 23. Plot of calcium ion binding as a function of pH 84 24. Plot of the amount of sialic acid released from factor II with neuraminidase as a function of time 86 25. Plot of the separation of neuraminidase and factor II on DEAE cellulose column 88 26. Plot of the elution profile obtained by passage of a mixture of factor II and a PC/PS dis­ persion incubated in the presence of calcium ions through Sephadex G-150 90 27. Plot of the elution profile from Sephadex G-150 of the products formed by treatment of prothrombin with thrombin 91 viii LIST OF ILLUSTRATIONS--Continued Figure Page 28. Plot of the molecular weight standard curve of cyanogen bromide peptides from fragment 1 determined by SDS electrophoresis 94 29. Elution profile of tryptic digestion mixture of fragment 1 96 30. Plot of the elution profile obtained by passage of a mixture of fragment 1 and a PC/PS dis­ persion incubated in the presence of calcium ions through Sephadex G-150 98 31. Plot of the elution of the noncalcium ion binding peptides after tryptic digestion of fragment 1 99 32.